Crops under cultivation suffer from many diseases caused by plant pathogenic fungi. One particularly damaging plant phytopathogenic fungus is Rhizoctonia solani which is widely distributed and causing common diseases of greenhouse-grown crops, field crops, vegetables, ornamentals, and fruits throughout the world. It also causes root rot and wilt disease of pyrethrum and geranium. Other detrimental fungal plant pathogens include Sclerotinia sclerotiorum, Thielavia basicola, Fusarium oxysporum, causing wilt, Pythium aphanidermatum causing lethal yellowing and damping off in numerous plants.
The incidence of various kinds of fungal diseases cause considerable damage to the medicinal and aromatic plants (MAPS) in different part of India. The occurrence in severe form may either kill emerging seedlings or reduce plant growth and adversely affect the crop yield. Among fungal pathogens, species of Rhizoctonia, Sclerotinia, Fusarium, Thielavia, Pythium, Helminthosporium, Curvularia, Alternaria and Colletotrichum are most important and common. They produce different kinds of diseases such as stem rot and twig blight (Sclerotinia sclerotiorum) on periwinkle, Egyptian henbane and Ammi majus; root rot & wilt (Rhizoctonia solani) diseases on pyrethrum, leaf blight (Curvularia andropogonis); lethal yellowing (Pythium aphanidermatum) and collar rot (Fusarium moniliforme) on Java citronella; damping-off (Pythium dissotocum), collar rot (Rhizoctonia solani) and leaf blight (Alternaria alternata) on opium poppy, stolon and root rot (Thielavia basicola), wilt (Fusarium oxysporum), leaf blight (Corynespora cassiicola) on mints and anthracnose (Colletotrichum acutatum) and wilt (Rhizoctonia solani) on geranium; (Alam et al 1983, Indian Phytopath. 367: 480–483; ibid 1992, Plant Disease 43:10578–1061; ibid 1994, Plant Pathology 43:1057–1061; ibid 1996, Indian Phytopath. 49:94–97; Sattar et al. 1993, Indian J. Plant Pathol 10: 10–11; ibid 1999, Indian J. Plant Pathol. 17:74–76; ibid 2002, J. Mycol. & Pl. Pathol. 32: 31–34).
The use of huge amount of fertilizers and chemical pesticides for maintaining the high productivity of crop has become fatal to human and animal health. They also poses many other serious problems including i) development of resistant strains of pathogen (Schwinn et al., (1991) “Control with Chemicals” Advances in Plant Pathology: vol. 7: Phytophtohora infestans, the Cause of Late Blight of Potato, Ingram et al., eds., Academic Press, 8: 255–266) ii) build up of harmful residues in the edible plant and iii) non-target side effect of beneficial micro flora. It is desirable to replace them with biopesticides derived from the microorganisms. They are as effective as broad-spectrum chemical pesticides, easily degradable and have low cost production. They are a distinct possibility for the future and can be successfully exploited in modern agriculture without affecting our precious ecosystem.
Plant growth promoting rhizobacteria (PGPR) exert a beneficial effect on the plant by causing plant growth promotion and/or suppressing plant pathogen population to avoid infection. Efforts to select and apply PGPR for control of specific soilbome fungal pathogens have been reviewed (Kloepper, 1993; Glick and Bashan, 1997 Biotechnology Advances 15, 353–378). In most of the cases, activity is due to production of metabolites such as antibiotics, hydrogen cyanide, iron-chelating siderophores, and cell wall-degrading enzymes, which directly inhibit the pathogen. Plant growth promotion by PGPR may also be an indirect mechanism leading to a reduction in the probability of a plant contracting a disease when the growth promotion results in shortening the time that a plant is in a susceptible state. An alternative mechanism for biological control by PGPR is by induced systemic resistance.
Bacillus subtilis and few other Bacillus spp. are used as biocontrol agents of fungal diseases caused by different plant pathogens (Swinburne et al. (1975) Trans. Brit. Mycol. Soc. 65:211–217, Baker et al. (1983) Phytopathology 73:1148–1152, Singh and Deverall, (1984) Trans. Br. Mycol. Soc. 83:487–490, Cook (1987) Proceedings Beltwide Cotton Production—Mechanization Research Conference, Cotton Council, Memphis, pp. 43–45, Gueldner, et al., (1988) J. Agric. Food Chem. 36:366–370, Pusey et al. (1988) Plant Dis. 72:622–626, Ferreira et al. (1991) Phytopathology 81:283–287, Sholberg et al. (1995) Can. J. Microbiol. 41:247–252, Asaka, and Shoda, (1996), Appl. Environ. Microbiol. 62:4081–4085). McKeen et al. (1986) Phytopathology 76:136–139 and Pusey and Robins (1991) U.S. Pat. No. 5,047,239 disclose control of post harvest fruit rot using B. subtilis. Among different Bacillus spp (B. subtilis, B. megaterium B. cereus, B. polymyxa and B. pumilus), B. subtilis is most exploited as biocontrol agent because it is considered to be a safe and potential biological control agent due to high thermal tolerance, rapid growth in liquid culture, ready formation of resistant spores. Handelsman (1991) U.S. Pat. No. 5,049,379 disclose that Zwittermicin-A producing B. cereus control damping off in alfalfa and soybeans by inhibiting root rot fungus. A Bacillus subtilis GBO3 strain is commercially used as seed dresser under the names KODIAC.™. HB. or GUS 2000.™. by Gustafson, Inc. Plano, Tex. 75093 (EPA Reg. No. 7501–146, 1992). This product is available as a 2.75% powder formulation containing not less than 5.5.times10. sup. 10 viable spores per gram and is to be applied at a rate ranging from 2–4 ounces per 100 pound of seed. The bacteria is said to colonize the developing root systems and compete with pathogens that would attack the roots. Huang et. al (1993), Can. J. Microbiol. 39: 227–233 investigated antagonistic behavior of two strains of Bacillus cereus; alf-87A & B-43 against Sclerotinia sclerotiorum, the causal agent of basal pod rot & end rot disease on dry pea. The vegetative growth & ascosporic germination of S. sclerotiorum are inhibited by diffusible metabolite produced by alf-87A but are unaffected by strain B-43 The spraying on pea plants at the pod development stage with alf-87A reduce the incidence of basal rot. The treatment of soybeans with B. cereus has been shown to improve soybean yield at field site (Osburn et al. 1995 Am. Phytopathol. Soc. 79: 551–556). Chen et al (2002) Chinese J. Biol. Control 18:45–46 report that antagonistic activity of B-916 strain of B. subtilis against R. solani is due to proteins because addition of ammonium sulphate in culture solution destroys its antagonistic ability. The application of B. subtilis reduced the stem canker disease caused by R. solani and common scab disease caused by Streptomyces scabies up to 63% and 70%, respectively. Liu et al. (1995) U.S. Pat. No. 5,403,583 disclosed a Bacillus sp., ATCC 55000 and a method to control the fungal plant pathogen, Rhizoctonia solani. Leifert et al. (1995), J. Appl Bacteriol. 78:97–108, reported the production of anti-Botrytis and anti-Alternaria antibiotics by two Bacillus strains, B. subtilis CL27 and B. pumilus CL 45. The whole broth and cell-free filtrates were active against Botrytis and Alternaria in in vitro tests and were active against Botrytis in in vivo small plant tests. Leifert et al. (1997) U.S. Pat. No. 5,597,565 disclosed that B. subtilis, B. pumilus and B. polymyxa are effective against post harvest disease caused by Alternaria brassicicola and Botrytis cinerea. They also disclose the presence of antibiotics produced in the cell-free culture filtrate and their activity at different pH values, but they do not identify these compounds. The compounds from B. subtilis lose activity at low pH, while the activity from the B. pumilus extracts occurs only at pH values below 5.6. Leifert, et al. (1998) U.S. Pat. No. 5,780,080 disclose that the growth of Botrytis cinerea and Alternaria brassicicola causing post-harvest disease is inhibited by applying isolates of Bacillus pumilus NCIMB 40489 and Bacillus subtilis NCIMB 40491 to cabbage at temperatures of about 20.degree C.
Bacilli are known to produce antifungal and antibacterial secondary metabolites (Korzybski, et al., 1978 “Section C: Antibiotic isolated from the genus Bacillus (Bacilliaceae)” In: Antibiotics—Origin, nature and properties, American Society for Microbiology, Washington D.C. Vol. III, pp. 1519–1661). The chemical nature of antibiotics produced by Bacillus spp. are peptide by the action of which they inhibit the growth of fungal plant pathogens in the microenvironment (Katz and Demain 1977 Bacteriological Reviews, 41, 449–474; Singh & Deveral 1984; McKeen et al. 1986; Utkhede et al (1986) Can. J. Microbiol. 32: 963–967; Wilson et al. (1989) Annual Review of Phytopathology. 27, 425–441, Hiraoka et al., (1992) J. Gen. Appl. Microbiol. 38:635–640.). Islam and Nandi (1985) J. Plant Dis. Protect. 92:241–246, disclose a Bacillus sp. with antagonism to Drechslera oryzae, the causal agent of rice brown spot. The same authors, Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):233–240, also disclose in-vitro antagonism of Bacillus sp. against Drechslera oryzae, Alternaria alternata and Fusarium roseum. They discussed three components in the culture filtrate. The most active antibiotic was highly soluble in water and methanol with a UV peak at 255 nm and a shoulder at 260 nm, that proved to be a polyoxin-like lipopeptide. Loeffler et al. (1986) J. Phytopathology 115:204–213, disclose B. subtilis, B. pumilus, B. licheniformis, and B. coagulans strains that produce various antibiotics with antifungal and antibacterial activity. B. pumilus produces bacilysin and iturin A. Bacilysin is a very small compound with a molecular weight of 270, that inhibits only yeast. The iturins, which are soluble in polar solvents, have broad antifungal and antibacterial activity. McKeen et al. (1986), have shown that antibiotics similar to the low molecular weight iturin cyclic polypeptides contribute to the fungicidal activity of B. subtilis. Rossall's (1991) U.S. Pat. No. 5,061,495 provides a novel antibiotic from B. subtilis that is 63,500 Dalton, precipitates at a pH below 5 and has activity against gram positive bacteria and fungi (Botrytis and Erysiphe). Rossall's (1994) U.S. Pat. No. 5,344,647 discloses Bacillus subtilis strains with broad anti-fungal activity. Stabb et al. (1994), Applied Environ. Microbiol. 60: 4404–4412 have identified different strains of B. subtilis, B. cereus, B. mycoides, B. thuringiensis that exhibit antifungal activity. These strains have been shown to produce zwittermicin-A and/or kanosamine (Milner et al. 1996, Applied Environ. Microbiol. 62: 3061–3066), that are effective against damping off disease caused by Phytophthora medicagenis, P. nicotianae, Pythium aphanidermatum or Sclerotinia minor. Zwittermicin-A is a water soluble, acid stable linear aminopolyol molecule (He et al. 1994, Tetrahedron Lett. 35: 2499–2502) with broad spectrum activity against many fungal and bacterial plant pathogens. Kanosaminealso inhibits a broad range of fungal plant pathogens and a few bacterial species (Milner et al. 1996).
Germida, et al. U.S. Pat. No. 6,015,553 disclosed Bacillus subtilis strain AQ743 that produces a metabolite exhibiting pesticidal activity against corn rootworm. Hassanein and El-Goorani (1992) J. Plant Pathol. 133: 239–246 reported that application of B. subtilis on wounded caster bean plants 30 min. before or simultaneously with inoculation of Agrobacterium tumefaciens, resulted in good control of crown gall without any phytotoxic injury or growth retarding side effect.
So in the present invention systematic experiments were planned to isolate and select superior strain of Bacillus strain for promoting the growth of medicinal and aromatic plants as well as inhibiting the growth of plant pathogenic fungi.